Vacuolar proton-translocating ATPases (V-ATPases) are found in all eukaryotic cells and appear to play both constitutive roles in all cells and more specialized roles in certain cell types. V-ATPases are multisubunit enzymes capable of coupling ATP hydrolysis to proton transport across membranes. The primary constitutive role of V-ATPases in all human cells appears to be acidification of certain intracellular compartments, and this acidification is critical for maintenance of the cell's internal organization and ability to respond to extracellular stimuli. Organelle acidification mediated by V-ATPases is exploited by a variety of pathogens, including certain viruses and toxins, to allow these pathogens to enter the cell cytoplasm; other pathogens manipulate V- ATPase activity to allow them to exist in intracellular compartments. V-ATPases play specialized roles in regulated secretory granules of neural cells, where they are involved in sequestration of neurotransmitters, and at the plasma membrane of kidney intercalated cells, osteoclasts, macrophages and neutrophils, where they are involved in urinary acidification, bone resorption, and regulation of cytoplasmic pH, respectively. The V-ATPase of the yeast Saccharomyces cerevisiae has proven to be an excellent experimental model for V-ATPases of other eukaryotes, including humans. The long-term goals of this research are to understand the structure, function, assembly, and regulation of the yeast V-ATPase. The specific aims of this proposal are directed toward understanding the interaction between the peripheral V1 sector of the V-ATPase, which is responsible for ATP hydrolysis, and the integral membrane Vo sector, which is responsible for proton transport. The interaction between the V1 and Vo sectors is central to the catalytic activity of V-ATPases and is also a major site of enzyme regulation. Toward this goal, the functions of two subunits (Vma5p and Vma13p) that are known to be important for interaction between the V1 and Vo sectors will be studied in detail, both in wild-type yeast cells and in strains containing point mutations in each subunit gene. Reversible dissociation of V1-Vo complexes into cytosolic V1 sectors and membrane- bound Vo sectors has been shown to occur in vivo in response to nutrient deprivation in yeast and in insect cells, and is probably a general mechanism of regulation. Cytosolic V1 sectors will be isolated from yeast cells, and the biochemical and enzymatic properties of these sectors will be examined. Links between catalytic activity, nucleotide binding, changes in cytosolic pH, and assembly state of the V-ATPase will be explored in biochemical studies, and the physiological benefits of dissociation of the V-ATPase under conditions of nutrient deprivation will be examined by isolating yeast mutants defective in disassembly of the enzyme.